1. Field of the Invention
This invention relates to a method for treating a subject having or suspected having a disease or condition associated with aberrant hypomethylation of one or more genes in the subject, the method involving a tailored epigenomic modification of the subject using methylated polynucleotides directed to hypomethylated genes in the subject. This invention also relates to a medicinal composition for treating a subject having or suspected of having a disease or condition associated with aberrant hypomethylation of one or more genes in the subject, the composition comprising methylated polynucleotides directed to hypomethylated genes in the subject, so as to result in a tailored epigenomic modification of the subject upon administration.
2. Description of the Related Art
Epigenetics is the study of somatic heritable regulations that modulate gene expression without altering the corresponding DNA sequence. The epigenetic regulation, found to be essential for all the cellular functions, includes a number of processes that modify DNA and histone structures, such as DNA methylation, histone modification and remodeling, as well as gene silencing by small RNAs (H. S. Cho et al. (2007), Journal of Biochemistry and Molecular Biology, 40:151-155). The onsets of diseases have been identified to be associated with abnormal epigenetic regulations, which are therefore proposed to be used as detection biomarkers and treatment targets of diseases associated therewith.
DNA methylation, the addition of a methyl group to the fifth carbon position of a cytosine residue by DNA methyltransferase (DNMT), occurs in CpG dinucleotides (A. Bird (2002), Genes Dev., 16:6-21) and is a key epigenetic feature of the human genome. These dinucleotides are usually distributed within stretches of 1- to 2-kb GC-rich DNA, named CpG islands, located in the promoter and/or first exon of 60% of human genes (A. Bird (2002), supra; M. Ehrlich (1982), Nucleic Acids Res., 10:2709-2721). Promoter methylation is known to participate in reorganizing chromatin structure and also plays a role in transcriptional inactivation (A. Bird (2002), supra; M. Ehrlich (2003), J. Cell Biochem., 88:899-910).
Studies have suggested that the CpG island in an active promoter is usually non-methylated, with the surrounding chromatin displaying an “open” configuration, allowing for the access of transcription factors and other co-activators to initiate gene expression (E. Ballestar and M. Esteller (2002), Carcinogenesis, 23:1103-1109; P. A. Jones and S. B. Baylin (2002), Nat. Rev. Genet., 3:415-428; K. P. Nephew and Tim H-M Huang (2003), Cancer Lett., 190:125-133). Furthermore, transcription factor occupancy may make the promoter inaccessible to repressors or other chromatin remodeling proteins. In contrast, the CpG islands in an inactive promoter may become methylated, with the associated chromatin exhibiting a “closed” configuration. As a result, the methylated area is no longer accessible to transcription factors, disabling the functional activity of the promoter (P. A. Jones and S. B. Baylin (2002), supra; S. B. Baylin et al. (2001), Hum. Mol. Genet., 10:687-692; M. R. Rountree et al. (2001), Oncogene, 20:3156-3165).
Two models have been offered to describe the molecular sequence leading to the establishment of epigenetic gene silencing. One model suggests that histone modifications are the primary initiating event in transient repression (H. Tamaru and E. U. Selker (2001), Nature (London), 414:277-283; H. Tamaru et al. (2003), Nat. Genet., 12:177-185). DNA methylation subsequently accumulates in the targeted CpG island, creating a heterochromatin environment to establish a heritable, long-term state of transcriptional silencing. However, a second model suggests that DNA methylation can actually specify unique histone codes for maintaining the silenced state of a gene (S. Eden et al. (1998), Nature (London), 394:842; D. Schubeler et al. (2000), Mol. Cell. Biol., 20:9103-9112; J. A. Fahmer et al. (2002), Cancer Res., 62:7213-7218; C. Stirzaker et al. (2004), Cancer Res., 64:3871-3877). Clearly, this epigenetic process is complex and multiple systems may be implemented for genes participating in different signaling pathways.
Accumulating evidence shows that epigenetic modifications have a crucial role in pathological disorders, including, for example, cancer, inherited diseases, and chronic inflammatory diseases. Abnormal DNA methylation is proposed to cause cancer in two major ways, one being the hypermethylation in certain genes especially the tumor suppressor genes, and the other being the global DNA demethylation.
For the treatment of diseases like cancer, chemical demethylation reagents or DNA methyltransferase (DNMT) inhibitors are proposed to restore the expression of tumor suppressor genes. The success in DNA demethylation and restoration of respective gene expression indicate that the epigenetic regulation like DNA methylation is more reversible and feasible than the genetic modification/therapy. While the demethylation agents are easier to be manipulated and bring the high hope for future clinical usage, they gain no control for what genes they are targeting, and uncontrolled demethylation may hurt the cell/individual instead. Therefore, targeted DNA methylation is one step closer for the epigenomic resolution of cancer.
WO 2006/113671 A2 discloses a method of treating a patient having prostate, lung, breast or colon cancer by either administering a demethylating agent to the patient in sufficient amounts to restore expression of a tumor-associated methylation silenced gene in the patient's tumor, or administering to the patient a polynucleotide encoding a polypeptide which is expressed in the patient's tumor thereby restoring expression of the polypeptide in the tumor.
WO 2007/008252 A1 discloses a method of treating cancer or inhibiting tumorigenesis, comprising (1) providing a subject who has, or is at risk for developing, cancer, wherein cells of the subject have a homozygous mutation at nucleotides 178 or 259 of the RB2/pl30 gene or have methylation of at least the region from about nucleotide +287 to about +411 of the RB2/pl30 gene; and (2) administering an effective amount of a demethylating agent to the subject. In addition, according to WO 2007/008252 A1, compounds such as siRNA and anti-ICBP90 antibodies, which inhibit ICBP90, pRb2/pl30 complex or DNA methylase proteins, can be used to treat cancer or inhibit proliferation of tumor cells.
While siRNAs cause gene silencing and are used to treat cancers, siRNAs are expensive and their constructions/designs are more time-consuming. The effective siRNA target sequences are usually different from the sequences that are used to profile the gene's expression. In other words, the profiling means can not be used directly for the interference of the cellular genome. Moreover, not all of the computed siRNA can successfully knockdown the related expression unless they have been tested.
On the other hand, methylated oligonucleotides have been synthesized and used to inhibit the expression of genes. For example, U.S. Pat. No. 5,840,497 issued to Robin Holliday (corresponding to WO 09429461 and EP 0707653 B1) discloses a method for the silencing of specific genes by DNA methylation. The method involves introducing into a cell a single-stranded oligonucleotide containing 5-methyl deoxycytosine, wherein the single-stranded oligonucleotide has a sequence complementary to a sequence within the promoter region of the gene to be silenced, and wherein the sequence within the promoter region contains at least one CpG doublet. In addition, in order to protect the single-stranded oligonucleotide from degradation by nucleases, it is preferred that methylated phosphorothioate oligonucleotides are used. While suggesting the use of methylated oligonucleotides in gene therapy or treatment of certain diseases, the disclosure of U.S. Pat. No. 5,840,497 fails to provide a convincing evidence verifying the effectiveness of methylated oligonucleotides in gene therapy or inherited human diseases.
WO 99/24560 (corresponding to U.S. Pat. No. 5,874,416) discloses a method of inhibiting the expression of a gene in a cell, comprising the step of administering to the cell a single-stranded oligonucleotide comprising nucleotide units wherein at least one cytosine of a cytosine-guanine base pair contains a methyl group in the 5 position of the cytosine nucleotide. In Specific Example 5 of the Specification of WO 99/24560, an in vivo experiment was performed to determine the effect of RZ1X (a 26-mer oligonucleotide with 5-MeC in each of its six CpG sites; see SEQ ID NO: 1) on cell tumorigenicity. However, in said in vivo experiment, MDA-MB 435 cells treated once with 20 mM RZ1X for 4 days were injected into nude mice for the development of tumor. As such, whether or not a methylated oligonucleotide like RZ1X is effective in the treatment of cancers still remains a question.
Diseases like cancer are polygenic and heterogenic in their nature. Therefore, disease themselves vary from person to person and from tissue to tissue. A previous report indicates that there are more than 60 mutations identified in any given breast tumor from any person and these mutations are distributed within 108 signaling pathways (L. D. Wood et al. (2007), Science, 318:1108-1113). They are not the mutations in p53 and APC with high occurrence frequency that cause the cancer, but the less frequent, accumulated mutations that really do. Several studies have reported that mutations should lead to more specific, patterned epigenomic mutations, and make the problem even more complicated (Y. W. Leu et al., (2004), Cancer Res., 64:8184-8192; R. Opavsky et al. (2007), PLoS Genetics, 3:1757-1769). The complexity of the causes of diseases may lead to a conclusion that it might not be feasible to treat a cancer by targeting a single gene/pathway. Based on this conclusion, the applicants tried to establish a linkage between epigenomic profiling and epigenomic modification so as to switch a cell's fate. Specifically, under the applicants' concept, epigenomic differences within the genome of a subject were comprehensively detected, and these detected differences were subsequently used as a means to conduct a tailored epigenomic modification upon the subject's genome.
Numerous reports indicate that estrogens and estrogen receptors (ERs), ERα and ERβ, play important roles in breast cancer genesis and progression, and tumor ER status is a critical determinant in breast cancer patients to elucidate response to adjuvant treatment with endocrine agent (Y. Kun et al. (2003), Hum. Mol. Genet., 12:3245-3258; R. Schiff et al. (2003), Clin. Cancer Res., 9:447s-454s). ERα functions as a ligand-inducible transcription factor that either up- or down-regulates transcription of various target genes by binding to downstream target gene promoter regions or tethering to other transcription factors, such as AP-1 and SP-1.
In a previous study, the applicants found 70 ERα target loci by the chromatin immunoprecipitation-on-chip (ChIP-on-chip) screening of about 9000 putative GC-rich promoter sequences (Y. W. Leu et al. (2004), supra). Methylation microarray analysis shows that progressive DNA methylation occurs in multiple ERα downstream targets (such as TRIP10, Kr-Znf1, and DCC) in breast cancer genomes. The applicants further used the ChIP-on-chip screening to identify the 70 ERα target loci and found that 27 loci of them were upregulated in ERα-positive (ERα+) human breast cancer cell line MCF-7 than in ERα-negative (ERα−) human breast cancer cell line MDA-MB-231 (V. X. Jin et al. (2004), Nucleic Acids Res., 32:6627-6635). It is well known that patients with ERα− breast cancer cells suffer greater drug resistance and metastasis, and have lower survival rate than those with ERα+ breast cancer cells.
To test the applicants' concept that detected epigenomic differences within the genome of a subject may be used to conduct a tailored epigenomic modification upon the subject's genome, in the present invention, the applicants conducted epigenomic profiling to determine ERα target genes which were hypomethylated in MDA-MB-231 cells than in MCF7 cells, followed by introducing into MDA-MB-231 cells methylated ERα targets that were synthesized based on the determined hypomethylated ERα target genes. It was found that both the in vitro and in vivo introductions of the methylated ERα targets elevated the methylation status of the ERα target genes in MDA-MB-231 cells, causing the death of MDA-MB-231 cells. Based on the obtained results, it is possible to treat a subject having or suspected of having a disease or condition associated with aberrant hypomethylation of one or more genes using methylated polynucleotides directed to the hypomethylated gene(s) present in the subject's genome.